Culex quinquefasciatus Say (Diptera: Culicidae) is one of the
dominant mosquito species in irrigated rice agro-ecosystems in Africa
(1,2) and has been linked with an increase in transmission and
prevalence of Bancroftian filariasis in these areas (3,4). Cx.
quinquefasciatus is also considered a potential vector of arboviruses
and an important pest species in irrigated rice agro-ecosystems (2).
Important factor promoting the abundance of Cx. quinquefasciatus in
these areas is its ability to utilise inundated rice fields for larval development (5). Proper understanding of the factors contributing to the
distribution and relative abundance of Cx. quinquefasciatus in rice
fields is therefore an important pre-requisite of vector control
operations. Unfortunately, little is known about the factors that
regulate spatio-temporal distribution of rice land mosquitoes. Where
studies have been done, the focus has mainly been on malaria vectors at
the expense of other species (6-8).

A large number of variables act interdependently to influence the
abundance of rice land mosquitoes. These include; physico-chemical and
biotic factors, quality and amount of available food, and several
agricultural practices. These parameters fluctuate greatly during the
course of rice growing cycle affecting distribution and abundance of
mosquito larvae. High algal productivity and associated photosynthesis
result in an increase in dissolved oxygen concentration favouring the
breeding of Cx. vishnui and their predators (9). The level of organic
matter and temperature of the water are important determinants of the
amount of food available for mosquito larvae (10, 11). An increase in
rice height has direct effect on some mosquito species by obstructing
gravid females from ovipositing and supporting greater diversity of
aquatic predators (12,13). Rice canopy cover may also reduce the amount
of sunlight reaching the water surface resulting in lower temperatures.
This in turn causes a decline in microbial growth upon which mosquito
larvae depend on and increases larval development time exposing them to
greater risks of contact with potential predators and competitors (14).

The use of agricultural inputs such as pesticides and fertilizers
also produce significant impact on larval abundance in rice fields.
Service (15) observed a resurgence of mosquito larvae after initial
control with insecticides due to slow rebounding of predator
populations. An increase in larval abundance has also been reported
after application of organic and inorganic fertilizers in rice fields.
In Madurai, India, the populations of Cx. vishnui, Cx. tritaeniorhynchus
and Cx. pseudovishnui (Diptera: Culicidae) increased in a dose-related
manner after application of inorganic nitrogen fertilizers (16). In
Mwea, Kenya, the populations of Anopheles arabiensis (Diptera:
Culicidae) and culicine larvae were significantly higher in ponds
treated with ammonium sulfate (AM) than in the control (7). Mogi (10)
observed a strong relationship between fertilizer application and
increased pupation rate.

Despite the important role of fertilizers in supporting high
populations of vector mosquitoes, little is known about the mechanisms
under which they act to regulate mosquito populations. Sunish and Reuben
(17) suggested that fertilizer application results in rapid
multiplication of microorganisms upon which mosquito larvae feed on.
Alternatively, ammonium sulfate has been suggested to act as an
oviposition attractant by reducing water turbidity (7). The objective of
the present study was to determine the effect of ammonium sulfate (AM)
and muriate of potash (MOP) fertilizers on survival and development of
immature stages of Cx. quinquefasciatus.

Material & Methods

The experiment was conducted using laboratory strain of Cx.
quinquefasciatus originally from Mwea Irrigation Scheme, Kenya
maintained at Medical Entomology Laboratory, University of Illinois at
Urbana-Champaign, USA. Within 6-12 h of egg hatch, 20 Cx.
quinquefasciatus larvae were transferred to larval pans (23 x 29 cm)
containing 0.422 g, 0.845 g, 1.2675 g and 1.690 g of either muriate of
potash or ammonium sulfate dissolved in one litre of deionised water.
These doses represented one-half, full dose, one and a half and twice
the doses used in Mwea rice fields, Kenya (50 kg/acre). A control was
included in which larvae were placed in one litre of deionised water
without any treatment. Each treatment was replicated five times and
therefore, the experiment included 45 larval pans containing 900 larvae.
Larval pans were maintained at 26[degrees]C and 70% RH throughout the
study. The larvae were fed with Tetramin fish food [Tetra Holding (U.S.)
Inc. Blacksburg, VA, USA] every other day up to III instar when rabbit
food was introduced. The pans were examined every 24 h and the number of
larvae of each instar present in each pan was recorded. To do this, an
observer, put his head close to the water surface and counted every
individual in the larval pan. Since the number of larvae in each pan
were only 20, it was possible to count each individual larva with
minimum error. It is only on very rare occasions that more larvae were
found in one census than in the previous census. In such cases, it was
assumed that the extra individual was present in the previous census.
Although it was not possible to state for certain the instar of every
individual, instar size classes were distinct enough that the majority
of individuals were categorised appropriately. In addition to larval
instars, individuals were also categorised as pupa and adult. The pupae
were transferred into larval cups containing the same kind of treatment
in which the larvae developed. Pupae from each replicate of a treatment
were placed in a cage and emerging adults were counted and recorded. The
experiment was terminated when all mosquito larvae had died or emerged.

Statistics: All statistical analyses were performed using SPSS version 11.5 (SPSS, Inc., Chicago, IL, USA). One way analysis of
variance was used to determine the effect of treatment on larval
development time, the number of individuals surviving in each life
stage, and the probability of surviving from one stage to the next.
Tukey's HSD test was further used to examine pairwise differences
among treatments in cases where a significant effect of treatment was
observed. The time taken for at least 50% of the larvae in a pan to
change to the next instar was taken as development period of that
particular instar and the mean for the five replicates gave the mean
development time.

Results

Results of ANOVA and Tukey's HSD tests revealed a strong
effect of treatment on the number of larvae, pupae and adults surviving
(Table 1). The numbers of I instar larvae surviving did not differ
significantly among treatments. However, the numbers of other larval
stages as well as the pupae and adults surviving were significantly
lower in AM treatments equal to or greater than 1.2675 g compared with
other treatments. Among the remaining treatments, survival of IV instar
larvae, pupae and adults was also significantly lower in treatments with
1.2675 g of MOP and 0.845 g of AM than in the remaining treatments. The
probability of survival from one larval stage to the next and from IV
instar to pupa was generally lower in treatments containing 1.2675 and
1.690 g of AM compared with the other treatments. The 0.845 g treatment
of AM had a similar effect as the higher dosages of the same treatment
on probability of survival of III to IV instar. In addition, the
probability of survival of II to III instar in this treatment was also
lower than the remaining treatments. The effect of treatment on
probability of survival from pupa to adult did not vary among treatments
(Table 1; and Figs. 1 & 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The development time of Cx. quinquefasciatus from I instar to adult
stage differed significantly among the different fertilizer treatments
(F = 33.025, df = 36, p < 0.001, Fig. 3). It took approximately six
days for larvae in the control to develop into adults and this was not
significantly different from seven days in the 0.422 and 0.845 g
treatments of muriate of potash. However, it took 2-3 days longer than
the control treatment for larvae to develop into adults in 1.2675 and
1.690 g treatments of MOP and 0.422 and 0.845 g treatments of AM. In
addition, it took 4 and 7 days longer than the control for the larvae in
1.690 and 1.2675 g of ammonium sulfate, respectively to complete
development.

Discussion

In the current study, fertilizer application had a significant
impact on survival and development of aquatic stages of Cx.
quinquefasciatus. These effects were more pronounced in AM treatments
than in the MOP treatments. None of the MOP dosages had significant
impact on survival of immatures of Cx. quinquefasciatus and only the
higher dosages showed significant impact on development time but in
significantly lower magnitudes compared with similar dosages of AM.
Ammonium sulfate accounted for up to 40% mortality rate and up to one
week delay in development time. Interestingly, the effects of 1.2675 and
1.690 g of AM on survival of Cx. quinquefasciatus immatures were
similar, contrary to the expected dose-dependent relationship. Moreover,
the larvae developed much slower in 1.2675 g than in 1.690 g of AM.
Because fertilizer application enhances multiplication of microorganisms
(18), it is likely that the higher dosage (1.690 g) of AM promoted
growth of microorganisms that were able to break down the fertilizers
thereby reducing their toxic effects. Also, the larvae may have acquired
the ability to compensate for the damages caused by higher dosages of
AM. Alternatively, the differences in dosages between the two groups
might have been too little to impact significantly on larval survival.
Further studies using widely spaced dosages could reveal more
information.

The mechanisms under which fertilizers act to impact larval
survival and development time is least known. At physiological level, it
is likely to involve ionic balance of the larvae. Considering that
fertilizer treatment did not affect the probability of survival from
pupae (non-feeding) to adults, it appears that consumption of treated
water together with food materials may have interfered with ionic
balance of the larvae resulting in reduced survival rate and increased
development time. The breakdown of ammonium sulfate into
N[H.sub.3.sup.+] and S[O.sub.4.sup.2+] could impact negatively on
mosquito larvae. At high level of internal concentration, ammonia is
known to interrupt nervous transmission (19) and this could result in
high mortality rate and increased development time. Exposure of two
snail species to 1-1.25 g/L doses of AM for 48 h resulted in 100%
mortality (20). Sulphur-coated urea has also been shown to be a possible
mosquito control agent in rice fields (21), suggesting that the sulphur
component of AM may partly have contributed to the observed mortality.
Higher levels of muriate of potash (KCl) may also affect mosquito
osmotic balance since [K.sup.+] and [Cl.sup.-] ions are the primary
constituents of urine in larval malpighian tubules (22).

Fertilizer application in rice fields has been reported to enhance
multiplication of microorganisms, which form the main diet of mosquito
larvae and in particular, ammonia nitrogen has been found to be an
oviposition attractant (18). Therefore, in contrast to the current
findings, fertilizer application in rice fields has been shown to
increase larval abundance (7) in a dose-related manner (16) and also to
accelerate pupation rates (10). However, it should be noted that in the
field, several factors may act interdependently to impact larval
abundance and survival, and fertilizer may be one of these factors (5,
17, 23). Fertilizer application in the rice fields is also applied
mostly by broadcasting and this result in heterogeneous distribution of
fertilizers and mosquitoes may have the ability to discriminate areas
with toxic dosages of fertilizers and avoid them. In addition, the rate
of fertilizer breakdown and utilisation may be greatly higher in the
rice fields because of presence of a wide range of biotic factors
including decomposition microorganisms, aquatic animals and vegetation
including rice plants. In contrast, the fertilizers used in this study
were scaled and dissolved in one litre of deionised water before the
larvae were introduced. Secondly, the study was restricted to larval
pans and only the food material was added. The rate of fertilizer
decomposition and utilisation may therefore have been much slower than
in the field, providing a toxic environment for mosquito larvae. Further
studies on the effect of fertilizers on survival and development of rice
land vector mosquitoes should be conducted with the aim of devising
control strategies based on farm managed practices.

Acknowledgement

This research was supported by NIH/NIAID grant # U01A1054889
(Robert Novak).

(15.) Service MW. Mortalities of the immature stages of species B
of Anopheles gambiae complex in Kenya: comparison between ricefields and
temporary pools, identification of predators, and effects of
insecticidal spraying. J Med Entomol 1977; 13: 535-45.